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Biomedical subjects

Jean-Paul Behr

Publications and source records attributed to Jean-Paul Behr.

At least 19 recordsLinked to original sources
PubMed2006

Cationic oligonucleotide-peptide conjugates with aggregating properties enter efficiently into cells while maintaining hybridization properties and enzymatic recognition.

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems. Moreover, stability of the duplex formed with the complementary sequence increased without inhibiting oligonucleotide enzyme recognition as shown by the properties of the conjugate to prime chain elongation by Taq DNA polymerase in a linear amplification/sequencing process.

Amino Acid Sequence↗
PubMed2006

Eradication of p53-mutated head and neck squamous cell carcinoma xenografts using nonviral p53 gene therapy and photochemical internalization.

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.

Animals↗
PubMed2006

omega-Hydrazino linear polyethylenimine: a monoconjugation building block for nucleic acid delivery.

Nonviral gene therapy requires efficient vectors that are able to deliver nucleic acids inside the targeted cell nucleus. Developing new tools for the synthesis of supramolecular vectors with improved transfection efficiency and better biodistribution is therefore a crucial issue. Here we describe the synthesis of a 140-mer linear polyethylenimine (L-PEI) terminated at one end by a highly nucleophilic hydrazine residue. This cationic polymer, whose backbone is well known for its remarkable gene-delivery efficiency, constitutes a building block for omega-regioselective conjugation to molecules through the formation of stable linkages such as the hydrazone bonds. To demonstrate the potential of the omega-hydrazino linear polyethylenimine, human serum transferrin, a ligand that is well know to improve gene-delivery systems, was used as a model of sensitive material. The blood protein was oxidized to generate an aldehyde function and was subsequently conjugated to hydrazino PEI. The new polyethylenimine-transferrin (PEI-Tf) vector was purified and was shown to condense plasmid DNA into compact superstructures compatible with cellular uptake. Finally, the cellular-binding and gene-delivery properties of PEI/DNA polyplexes incorporating different quantities of transferrin were evaluated by FACS analysis and luciferase assay.

DNA↗
PubMed2005

Monomolecular DNA nanoparticles for intravenous delivery of genes.

Delivery is the major obstacle to success of nucleic-acid-based therapies. We have neutralized DNA with a cationic detergent (C12CCP) obtained by amide bond formation between dodecanoic acid, cysteinyl-cysteine, and diaminopropane. Subsequent detergent polymerization by formation of intermolecular disulfide bonds within the condensed plasmid DNA leads to 32-nm-large neutral particles. (C12CCP)n/DNA complexes are more stable than those formed with other gene delivery agents toward exchange with extracellular polyanions such as glycosaminoglycans. Yet exposure to phosphatidylserine, an ubiquitous intracellular anionic lipid, still releases DNA from the complexes for transcription of the carried gene. Pharmacokinetics and biodistribution in mice showed that 25% of the complexes were still circulating after 30 min (2% for other cationic lipid vectors) in a form essentially not bound to blood cells. Altogether, straightforward control over size and surface charge, stability toward aggregation or exchange, and favorable pharmacokinetics make these complexes attractive vehicles for reaching tumor metastases after injection in the blood circulation.

Animals↗
PubMed2005

Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels.

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.

Analysis of Variance↗
PubMed2005

Polyethylenimine (PEI).

Since the first edition of this book in 1999 the field of gene therapy has been the arena both for major advances that justified the early hopes placed in the concept, and for ever-present impatience with the slowness of overall progress. On the positive side, gene therapy obtained its first brilliant success, though not where most efforts were invested and not with a synthetic vector (Cavazzana-Calvo et al., 2000). Yet the search for efficient molecules is still very active, in part because the negative consequences of using viral vectors somewhat shadow the brilliant picture (Hacein-Bey-Abina et al., 2003).

Journal Article↗
PubMed2005

DermaVir: a novel topical vaccine for HIV/AIDS.

Human immunodeficiency virus (HIV) vaccines have the potential to improve antiretroviral drug treatment by inducing cytotoxic killing of HIV-infected cells. Prophylactic vaccines utilize new antigens to initiate immunity; however, in HIV-infected individuals the load of viral antigen is not the limiting factor for the restoration of immune responses. Here we describe a novel immunization strategy with DermaVir that improves viral antigen presentation using dendritic cells (DC). DermaVir contains a distinctive plasmid DNA expressing all HIV proteins except integrase to induce immune responses with broad specificity. The DNA is formulated to a mannosilated particle to target antigen-presenting cells and to protect the DNA from intracellular degradation. After topical application, DermaVir-transduced cells migrate from the skin to the draining lymph node and interdigitate as DermaVir-expressing, antigen-presenting DC. We compared the immunogenicity of topical and ex vivo DC-based DermaVir vaccinations in naive rhesus macaques. Both vaccinations induced simian immunodeficiency virus-specific CD4 helper and CD8 memory T cells detected by an in vivo skin test and an in vitro intracellular cytokine-based assay. Topical DermaVir vaccination represents an improvement upon existing ex vivo DC-based immunization technologies and may provide a new therapeutic option for HIV-infected patients.

AIDS Vaccines↗
PubMed2005

Targeted gene delivery to cancer cells with nanometric DNA particles enveloped with folic acid using a polymerisable anchor.

Progress in the design of gene delivery system is vital for cancer gene therapy since many physiological and intracellular barriers remain. We have developed a technology for condensing genes into nanometric delivery systems. In this paper, we present a novel strategy for decorating 30 nm DNA particles with folic acid for cancer cell recognition. Physicochemical and biological experiments show that these DNA complexes selectively bind to cells expressing the corresponding folic acid receptor.

Carrier Proteins↗
PubMed2005

Polyethylenimine (PEI).

Since the first edition of this book in 1999 the field of gene therapy has been the arena both for major advances that justified the early hopes placed in the concept, and for ever-present impatience with the slowness of overall progress. On the positive side, gene therapy obtained its first brilliant success, though not where most efforts were invested and not with a synthetic vector (Cavazzana-Calvo et al., 2000). Yet the search for efficient molecules is still very active, in part because the negative consequences of using viral vectors somewhat shadow the brilliant picture (Hacein-Bey-Abina et al., 2003). Clinical trial reports using the first generation of non-viral vectors, that is, cationic lipids, emphasize safety more than efficacy. The next generation, namely cationic polymers, is coming to maturity. This is illustrated in Fig. 8.1 by a literature survey that compares the most used "open source" vector in each category, the lipid DOTAP and polymer PEI. This "coming of age" is also highlighted by the number of planned clinical trials using PEI, trials we shall discuss in the last paragraph of this chapter. Advances have not only been made toward therapy, but also toward a better understanding of the mechanisms underlying gene delivery. Finally, PEI has become one of the most popular reagents for transfection of cells in culture, on a fast-growing market that is boosted by human and animal genome sequencing.

Central Nervous System↗
PubMed2004

The cyclin-dependent kinase inhibitor p21(cip1/waf1) enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells.

Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy.

Adenocarcinoma↗
PubMed2004

Effective polyethylenimine-mediated gene transfer into human endothelial cells.

BACKGROUND: The major advantage in choosing non-viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases. METHODS: Efficacies of a 22-kDa linear PEI (L-PEI) and its glucose-grafted derivative (L-PEI-Glc(4)) were compared for gene transfer into human umbilical vein endothelial cells (HUVEC) using the reporter gene luciferase. Cells were incubated for 2, 4 and 24 h with PEI/DNA complexes made in 150 mM sodium chloride (NaCl) or in 5% glucose solution. Luciferase activity was measured 24 h after the onset of transfection. The effects of low (2%) and high (30%) concentrations of serum on transfection efficacy were assessed as well. We then studied the intracellular fate of the PEI/DNA complexes labelled with the DNA intercalator YOYO-1 using flow cytometry analysis (FACS) and confocal microscopy. RESULTS: PEI/DNA complexes formed in NaCl led to a higher transfection efficacy than those made in glucose. The optimal formulation, depending on the incubation time and the presence of serum in the medium, was obtained using DNA complexed to L-PEI-Glc(4) and incubated for 4 h with the cells. This condition led to 50% fluorescent cells after GFP transfection. A high serum concentration diminished the L-PEI associated toxicity but decreased L-PEI-Glc(4) transfection efficiency. FACS analysis using both vectors showed that almost 90% of the cells had internalized the DNA complexes. Confocal microscopic observations showed a fast attachment of the complexes to the cell surface followed by inclusion into vesicles that migrated to the perinuclear region. CONCLUSIONS: In this work, we defined the optimal conditions for gene delivery in HUVEC. These conditions were obtained when using derivatives L-PEI and L-PEI-Glc(4) complexed with DNA in 150 mM NaCl and added to cells for 2 and 4 h, respectively. Cellular trafficking of the complexes suggested that cell entry was not a limiting factor for gene delivery using PEI. This study underlined the interest in PEIs as efficient vectors for gene transfer into human endothelial cells.

DNA↗
PubMed2004

A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin.

BACKGROUND: Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface. Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells. Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles. METHODS: We propose a model for cell entry involving syndecans as receptors for the DNA complexes by comparing transfection with bacteria uptake and using drug inhibition experiments along with confocal microscopy. RESULTS: When combined with results from the literature, our data suggest the following sequence of events: after initial particle binding, gradual electrostatic zippering of the plasma membrane onto the particle is sustained by lateral diffusion of syndecan molecules that cluster into cholesterol-rich rafts. Clustering in turn triggers PKC activity and linker protein-mediated actin binding to the cytoplasmic tail of the syndecans. Resulting tension fibers and a growing network of cortical actin may then pull the particle into the cell. CONCLUSIONS: Diversion of integrin- and syndecan-mediated cell adhesion processes for particle engulfment appears to be widely exploited by animals (chylomicrons), by pathogens (bacteria, viruses) and, as suggested here, by non-viral vectors.

Actins↗
PubMed2004

Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI-DNA complexes.

BACKGROUND: p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild-type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy. METHODS: p53 nonviral-mediated gene transfer was achieved using glucosylated polyethylenimine (PEI) in conjunction with photochemical internalisation (PCI). Experimental conditions were optimised using the green fluorescent protein (GFP) as a reporter. p53 gene transfer was then evaluated using semi-quantitative RT-PCR in p53-deleted PANC3 and p53-mutated FaDu cell lines. Following gene transfer, induction of apoptosis was investigated using phosphatidylserine externalisation and nuclear fragmentation assays. Induction of long-term cell death was analysed using colony-forming assays. RESULTS: PCI was found to enhance GFP gene transfer after 48 h in both cell lines. Whether using glucosylated-PEI alone or associated with PCI, p53 gene transfer was achieved with subsequent recovery of p53 mRNA expression in PANC3 cells and a significant 4-fold increase in p53 mRNA expression in FaDu cells. PCI was found to further enhance p53 mRNA expression by 2.3-fold in PANC3 cells. Spontaneous induction of apoptosis following wt-p53 gene transfer was achieved in both cell lines. PCI was found to enhance apoptosis up to levels similar to those achieved with chemotherapy. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used in both cell lines, yielding up to 60% cell death. CONCLUSIONS: PCI increases glucosylated-PEI-mediated p53 gene transfer, apoptosis as well as cell death in mutant p53 human cancer cells.

Apoptosis↗
PubMed2004

Intracytoplasmic delivery of anionic proteins.

Protein delivery is emerging as an interesting alternative to gene delivery. We have used our experience with transfection to develop a technique for efficient delivery of anionic proteins such as antibodies into the cytoplasm of cells. As for DNA, when complexed with cationic lipids, large amounts of proteins are shown to enter adherent cells via ubiquitously expressed syndecans. However, protein surface area rather than electric charge ratio governs the delivery characteristics. Delivery of anti-beta-actin and anti-alpha-tubulin IgG's leads to fiber depolymerization. Intracellular delivery of an antibody could thus be regarded as another method for interfering with gene activity.

Animals↗
PubMed2004

Genuine DNA/polyethylenimine (PEI) complexes improve transfection properties and cell survival.

Polyethylenimine (PEI) has been described as one of the most efficient cationic polymers for in vitro gene delivery. Systemic delivery of PEI/DNA polyplexes leads to a lung-expression tropism. Selective in vivo gene transfer would require targeting and stealth particles. Here, we describe two strategies for chemically coupling polyethylene glycol (PEG) to PEI, to form protected ligand-bearing particles. Pre-grafted PEG-PEI polymers lost their DNA condensing property, hence their poor performances. Coupling PEG to pre-formed PEI/DNA particles led to the expected physical properties. However, low transfection efficacies raised the question of the fate of excess free polymer in solution. We have developed a straightforward a purification assay, which uses centrifugation-based ultrafiltration. Crude polyplexes were purified, with up to 60% of the initial PEI dose being removed. The resulting purified and unshielded PEI/DNA polyplexes are more efficient for transfection and less toxic to cells in culture than the crude ones. Moreover, the in vivo toxicity of the polyplexes was greatly reduced, without affecting their efficacy.

Animals↗
PubMed2004

Towards multifunctional synthetic vectors.

Recent progress on the fronts of design, formulation and delivery with synthetic vectors has increased the potential of non-viral approaches to attain therapeutic applications. However, a number of hurdles remain to be cleared. On the one hand, we have the problem of producing and formulating stable, diffusible complexes and on the other hand, we have to face the fact that in vivo delivery is probably too complex and multifaceted to be achieved successfully with a single carrier molecule in most instances. This review concentrates on this latter aspect and the design of artificial multi-component vectors aimed at providing solutions to membrane crossing, endosomal escape and navigation through the nuclear pore. In many instances the solutions proposed have been inspired by natural mechanisms exploited by bacteria and viruses. However, entirely novel chemical approaches such as monomolecular DNA condensation via detergent dimerization, or endosome disruption by osmotic swelling, are also being investigated and developed. The combination of these naturally inspired and chemically-originated approaches is bringing us continually closer to the concept of constructing an artificial virus capable of delivering viable nucleic acid-based pharmaceuticals to defined cells in vivo. This review considers the most successful current solutions to the main biological barriers to gene delivery including appropriate DNA compaction, cell targeting and entry, vacuole escape, nuclear import and in vivo delivery. In the latter section, emphasis is placed on one of the most versatile non-viral vectors currently available, linear polyethylenimine.

Active Transport, Cell Nucleus↗